Squaric acid activated carrier usable for immobilization of compounds containing amine groups

ABSTRACT

A storage stable composition comprising a carrier usable for the immobilization of compounds containing an amine group, for instance biomolecules and/or affinity ligands. The carrier has been activated to exhibit a squaric acid derivative linked to the carrier and is placed in contact with an aqueous medium. A kit comprising the storage stable composition is also disclosed.

This application is a 371 of PCT/SE99/01861 filed Oct. 14, 1999.

The present invention relates to storage stable, activated carrier inthe form of matrices including surfaces. In particular it relates tosuch matrices suitable as carrier to which compounds containing an aminegroup may be covalently bound.

The invention also relates to a kit comprising such activated carrier,the kit being shippable to customers by virtue of the long termstability of said carrier.

In the context of this description of the invention the wordscarrier/carriers, matrix/matrices and medium/media are usedinterchangeable, if not otherwise said.

BACKGROUND OF THE INVENTION

The immobilization of biomolecules on various carriers finds greatutility in research and industry, and many methods have been disclosedin the literature. These methods comprise an activation of a carrier,e.g. a matrix in the form of a gel, a surface of a polymer bead etc. Theactivation process generally comprises subjecting the carrier to areactive agent that reacts with —OH groups or amine groups on thecarrier, thereby, either directly or subsequent to further steps,forming intermediate reactive structures/sites. These structures maythen be reacted with the compound in question, thereby immobilizing thecompound.

Illustrative examples of such methods are the CNBr method, and epoxyactivation. The activated intermediates obtained by these methods mustbe freeze dried in order to be shippable to customers. Freeze drying isfairly complicated and in addition require processing at the customerbefore the freeze dried intermediates can be used.

These and other activation and immobilization methods are described in“Immobilized affinity ligand techniques” by G. T. Hermanson, AcademicPress, 1992.

In WO 95/15983 (Glüsenkamp et al) there is disclosed a process forimmobilizing biomolecules and affinity ligands on carriers. This processcomprises reacting an aminated polymer carrier with a bifunctionalelectrophilic squaric acid derivative as activating agents to form anactivated intermediate (activated matrix or activated media or activatedcarrier), and coupling a biomolecule to the activated carrier.

For most of the above-mentioned activation processes it has beencommonly known that the activated carriers are relatively unstabletowards hydrolysis. Remembering that commercially available activatedcarrier media should have as long shelf time as possible, for instancefrom one up to several months, it is no surprise that commercialproducts comprising activated matrices typically have been sold in formof lyophilized compositions. For squarate activated carrier media we arenot aware of any commercial products differing from what has been saidabove. When looking into the scientific literature it appears that mostsynthetic routes in which squarate activated material are obtainedtypically end up in crystallized and/or dried material. In thealternative the activated material is used more or less directly uponsynthesis. See for instance Kamath et al., Glycoconjugate J. 13 (1996)315-319; and Ola Blixt, Thesis: Enzymatic solid phase synthesis ofcarbohydrates, Swedish University of Agricultural Sciences, Uppsala,Sweden (1999) (see in particular page 50); DE 4341524 (Glüsenkamp etal); and DE 19624990 (Glësenkamp et al). An investigation of thehydrolysis of soluble squarate activated materials (monoamides) has beenpublished (Glësenkamp et al., Z. Naturforsch. 46c (1991) 498-501). Thestorage of squarate activated aminated matrices moistured with methanolor in ethanol atmosphere has been described in the experimental part ofWO. 9515983 (Glësenkamp et al). The reactivity is expected to beretained during these storing conditions because reaction with methanoland ethanol, respectively, will only mean a transesterificationretaining the ester status of the activated group and thus alsomaintaining its reactivity.

It would be advantageous to provide a commercial product in form of anactivated carrier comprising reactive squaryl groups said activatedcarrier being dispensed in aqueous media and having a shelf life of from1-2 months up to several months without showing any significant loss inactivity. This is in particular true in case the use is for immobilizingwater-soluble compounds to the activated medium, for instanceamine-containing biomolecules such as proteins. The aqueous media istypically an aqueous liquid.

It would also be advantageous in case such storage stable compositionswould be resistant towards microbial growth.

Other publications connected to squarate activated materials are Tietzeet al., Chem. Ber. 124(5) (1991) 1215-1221; Tietze et al., BioconjugateChem 2 (1991) 148-153; Hällgren et al., J. Carbohydrate Chem. 14(4&5)(1995) 453-46; Ausanneau et al., Bioorganic & Medicinal Chemistry 4(1996) 2003-2010; and Pozsgay et al., J. Org. Chem. 62 (1997) 2832-2846.

SUMMARY OF THE INVENTION

The object of the present invention is therefore to provide a storagestable activated carrier comprising reactive squaryl groups, saidcarrier being dispersed in an aqueous medium for use in theimmobilization of nucleophilic compounds, typically amine containingcompounds. The activated medium should be stable during storage and/orresistent against bacterial growth over extended periods of time.

For the purpose of the invention the term “activated medium” or“activated carrier” or “activated matrix” mean a carrier to be used as acarrier for a nucleophilic compound. Said carrier has been provided withreactive squaryl groups enabling easy coupling of said compound to saidcarrier. The activated carrier should be regarded as an intermediate inthe process of making immobilized forms of said compounds.

Nucleophilic compounds that are contemplated in the context of theinvention typically contain a primary and/or a secondary amine group,for instance proteins or other compounds exhibiting peptide structure orother biomolecules and other organic compounds exhibiting this type ofamine groups.

The inventor has now surprisingly discovered that a carrier as definedabove comprising amine groups activated with an electrophilic squaricacid derivative may be stable in aqueous media. Unexpectedly it has beenrecognized that the rate of hydrolysis of reactive squarate groupsbound; to carriers is sufficiently slow for enabling storage of theactivated carriers in aqueous media.

Thus, in accordance with the present invention there is provided astorage stable composition comprising a carrier as defined above andusable for the immobilization of nucleophilic compounds. The carriercomprises primary or secondary amino groups that have been activated tocarry reactive squaric acid derived groups linked to said primary orsecondary amine sites on said carrier, said carrier being in contactwith an aqueous medium, e.g. an aqueous liquid.

In a second aspect of the invention there is provided a method ofpreparing such a composition. This method is defined in claim 11.

Furthermore, in accordance with the invention there is provided a kitfor storing the composition over extended periods of time, this beingdefined in claim 10.

The composition according to the present invention is stable for anextended period of time as defined above, for instance for a week, amonth or even longer periods of time, such as two, three or more months.

Preferred embodiments are defined in the dependent claims.

DETAILED DESCRIPTION OF THE INVENTION

The squaric acid derivatives useful for making the activated carriersmay be selected among bifunctional electrophilic squaric acidderivatives, such as dialkoxyesters (e.g. dimethylester, diethylester,dibutylester and also corresponding mixed diester (squarates)),dihalides and diimidazolides. Other reactive derivatives may also beused. The activation reaction is normally run in inert media. SeeGlüsenkamp WO9515983. In case the reactive group formed on the carrieris too reactive to be stored in the contemplated aqueous media, it maybe easily transformed to a group of lower reactivity. For instance, incase a squaric acid dihalide is used to prepare the activated carriers,the formed monohalide group may be transformed to the correspondingester function, for instance by running the reaction in the presence ofan alcohol.

A reactive intermediate according to the invention may be represented bythe general formula

wherein

represents a matrix (carrier) containing primary and/or secondary aminegroups binding to the squaric acid derived structure (squaryl group,e.g. squarate structure) which in turn constitutes the reactive group inthe formula above. There are often several reactive squaric acid derivedgroups (e.g. squarate groups) on one and the same matrix P.

A and B independently of each other can be O or S;

X can be O or S; and

R may represent an inert organic group that together with X and thesquaryl group forms an electrophilic structure.

R is, for instance, a C₁ to C₃₀ hydrocarbon group comprising one or morestraight, branched or cyclic hydrocarbon chains that may be broken atone or more locations by a thioether sulphur or an ether oxygen and/orsubstituted with one or more R′O- groups where R′ is hydrogen and/or C₁to C₆ alkyl or corresponding sulphur analogues (R′S-). The preferred Rsare C₁ to C₆ alkyl, such as methyl, ethyl, n-propyl, isopropyl, andpossibly also isoforms of butyl. Preferably there are at most one O or Satom on each carbon atom in R.

R may also be an imidazolide group or another group resulting in thesquarate structure in the general formula being a reactive electrophilicsquarate group on the carrier. In this case the stability of theactivated carrier shall be comparable to the stability of the squaricacid mono amide mono alkyl ester groups defined above (R=alkyl).

By the term “inert” is meant that R does not contain any group thatadversely affect the reactivity of the activated squaryl group shown.Destabilizing groups in R may for instance be primary and secondaryamines.

By the term “stable” is meant that the ability to react with primary andsecondary amines is retained for a prolonged time without beingsignificantly or irreproducibly decreased. This in turns means that theactivated intermediate can be stable even if the activated intermediateparticipate in transesterifications with alcohol groups present in thestorage media, the carrier or the inert group R.

As carriers to be activated with squaric acid derivatives defined above,matrices exhibiting primary and/or secondary amine groups can be used.

The term “matrix” is to be understood to encompass any physical formsuch as particles, monoliths, fibers, membranes, tube walls,capillaries, surfaces etc. The matrix may be porous or non-porous andsoluble, insoluble or insolubilizable in aqueous media or other liquids.

In the preferred modes of the invention the matrix is based on a polymerthat exposes a hydrophilic surface to the aqueous media used, i.e.expose hydroxy (—OH), carboxy (—COOH), carboxamido (—CONH₂, possibly inN-substituted forms), amino (—NH2, possibly in substituted form), oligo-or polyethylenoxy groups on their external and, if present, also oninternal surfaces. Typically the matrix is of the same kind as thosenormally used as chromatographic matrices. The polymer may, forinstance, be based on polysaccharides, such as dextran, starch,cellulose, pullulan, agarose etc., which if necessary have beencross-linked, for instance with bisepoxides, epihalohydrins,1,2,3-trihalo substituted lower hydrocarbons, to provide a suitableporosity and rigidity. The matrix may also be based on syntheticpolymers, such as polyvinyl alcohol, poly hydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, poly acrylamides, polymethacrylamides etc.In case of hydrophobic polymers, such as those based on divinyl andmonovinyl substituted benzenes, the surfaces of the matrix are oftenhydrophilized to expose hydrophilic groups as defined above to asurrounding aqueous liquid.

The matrices may also be of inorganic nature, e.g. silica, zirconiumoxide etc.

If the matrices as such do not exhibit amine functionality they may bederivatized by methods known by the man skilled in the art to do so.

The activating method is fully described in the referencedWO-publication, and will not be discussed herein.

The storage medium for the intermediates in question is aqueous,typically in form of an aqueous liquid. It preferably comprises anantibacterially active component. This component preferably is selectedform organic solvents suitably miscible with water.

A preferred class of components is alcohols, in particular alkanols,such as methanol, ethanol, propanol, iso-propanol, butanol, sec-butanol,tert-butanol. The most preferred alcohol is ethanol.

The amount of the antibacterial component needed is an amount sufficientfor preventing bacterial or microbial growth in the composition.

The composition of the aqueous medium may comprise water in an amount of5-100%, preferably 20-95%, more preferably 40-85%. The amount of organicsolvent suitably miscible with water is in the interval 5-95%,preferably 5-80%, more preferably 10-60% such as 25-60 . Volume-% (v/v)is contemplated. The organic solvent is preferably a water-misciblealcohol.

The pH of the aqueous medium may be in the range 5-9. Standard buffersystems that do not adversely affect the stability may be used, e.g. 0.3M borate buffer. Amine buffers, in particular adjusted to pH below 1minus their pKa should be avoided.

When being used the activated carrier is contacted with an organiccompound containing an amine group under conditions permitting bindingof the amine compound to the carrier via a newly formed squarate amidestructure. The compound containing the amine function is preferably abioorganic molecule. Typically the amine compound is a member of aso-called affinity pair. Examples of affinity pairs areantigen/hapten—antibody, carbohydrate structure—lectin, complementarynucleic acid sequences and any other native or synthetic ligand-receptorpair in which the ligand and receptor are able to cling to each othervia affinity. One special class of affinity pairs has biological origin.They are consequently called bioaffinity pairs.

BRIEF DESCRIPTION OF DRAWING

The invention will now be described in detail by way of examples andwith reference to Scheme A and FIG. 1.

In scheme A, RNH₂ is the compound to be immobilized to the matrix. Thescheme is focusing on one kind of introduced amino groups. The reactionwill in principle be the same for the other amino groups.

FIG. 1 gives a graph showing the long-term stability of a compositionaccording to the invention. The amino acid analysis data showncorrespond to coupling yields of 75-85%. We have immobilized otherligands with similar or higher yields.

EXAMPLE 1 Preparation of Amine Containing Beads

Polymer beads (Sephacryl S-200, Amersham Pharmacia Biotech AB, Uppsala,Sweden) were amine functionalized in the following way.

Epoxy activated beads were washed with water and drained, and then addedto a reaction vessel and an equal volume of 1 M NH₃ was added to thevessel. The vessel was sealed and circle shaken in a water bath at 50°C. for 23 h. The beads were washed with water (>10×gel volume) andstored in 20% ethanol in water at +2−+8° C.

After amination the beads will contain different amino groups—CH₂CHOHCH₂NH₂ (obtained by reaction of ammonia with epoxy groups),—NHCOCH₂CHNH₂ (obtained by reaction of ammonia with acrylamido groupsand corresponding crosslinking groups obtained via further reaction withepoxy and acrylamido groups.

EXAMPLE 2 Activation of Aminated Matrix with 3,4-Diethoxy-squarate

The amine functionalized Sephacryl (100 ml, from Example 1) stored in20% EtOH, was washed with absolute ethanol (10×1 gel volumes). The beadswere added to a flask with screw cap. An equal volume of absoluteethanol was added followed with benzyl alcohol (ca 250 μl/50 ml ethanol)and the squarate (ca 1.5-2 equivalents compared to the amine contents ofthe beads). The reagents were mixed and a small sample of the liquidphase, used as reference for HPLC, was taken out immediately. The flaskwas shaken in a water-bath at 20-60 ° C. The progress of the reactionwas followed by HPLC by taking out small samples of the liquid phase,after regular intervals, dilute 50 times with EtOH (30 μl in 1.5 ml) andanalyze on a Shimadzu HPLC. Benzyl alcohol was used as an internalstandard in the HPLC analysis. The substitution degree was assumed to beapproximately equal to the consumed amount of the squarate reagent.

The beads were washed with ethanol 99.5% followed with 20% ethanol. Theactivated beads were divided in 10 ml samples slurried in 20% ethanoland stored at 2-8° C.

EXAMPLE 3 Coupling of Porcine Trypsin

The activated beads, stored in 20% EtOH, were washed with water (ca 5×1gel volume, gv), followed with a borate buffer 0.3M borate 1.5 M Na2SO₄,pH 9. The enzyme solution was made by dissolving 100 mg Porcine trypsinin ca 10 ml 0.5 M Na2SO4 and 0.3 M borate buffer pH 9 which was added tothe beads The mixture was shaken at room temperature for 6 hrs. Thebeads were washed with tris buffer (0.1 M, 0.5 M NaCl, pH 8) 4×1 gelvolumes. Followed, alternatively four times with tris and acetate buffer(0.1 M, pH 5), and finally with water five times. Stored in 20% EtOH.

EXAMPLE 4 Stability Test

Stability tests on the intermediates in the storage mode according tothe present invention (the storage medium used in the examples is 20%ethanol in water, temperature 2-8° C.) were made by taking samples ofthe intermediates at irregular intervals during 66 weeks (more preciselyat 1, 6, 10, 12, 18, 26, 40 and 66 weeks).

These samples were used for immobilization of porcine trypsin on thematrix according to example 3. The substitution degree was investigatedby amino acid analysis.

FIG. 1 shows the results from the storage test. From this figure it canbe concluded that within the experimental errors, the intermediates arestable in the aqueous storage medium over an extended period of time,i.e. at least about 66 weeks.

The composition according to the invention is suitably prepared byreplacing the liquid reaction medium from the activated carrier medium,for instance by washing, with the aqueous medium that ultimately will beused for storing the composition.

The composition is delivered to the customer as a kit comprising astorage container containing the composition, i.e. the activated mediumin contact with the aqueous medium. Such storage means can comprisebottles, ampoules, flasks, and other closed vessels not permittingevaporation. In case of carrier media in the form of open surfaces (e.g.wells of a microtiter plate), and monolithic porous and nonporousmatrices, strips and the like, the medium itself may function as thestorage means, provided there is an appropriate cover means preventingevaporation from the activated surface.

What is claimed is:
 1. A storage stable composition comprising a carriermedium exhibiting primary and secondary amine groups and usable for theimmobilization of amine group containing compounds, said carrier mediumbeing activated to exhibit electrophilic squaric acid derived groupslinked to said medium, said carrier medium being in contact with anaqueous medium, said composition being comprised in a closed vessel notpermitting evaporation of said aqueous medium.
 2. The composition ofclaim 1, wherein said activated carrier medium is represented by theformula

wherein P represents said carrier; A and B independently of each othercan be O or S; X can be O or S; and R represents and inert organic groupthat together with X and the squaryl group forms an electrophilicstructure.
 3. The composition of claim 1, further comprising anantibacterially active component.
 4. The composition of claim 3, whereinthe antibacterially active component is a solvent miscible with water.5. The composition of claim 1, wherein said aqueous medium compriseswater in an amount of 5-100%.
 6. The composition of claim 1, whereinsaid aqueous medium comprises an alcohol in an amount of 5-95%.
 7. Thecomposition of claim 1, which is stable for at least one week.
 8. Thecomposition of claim 1, wherein said carrier medium is in the form ofbeads.
 9. The composition of claim 1, wherein the pH of the aqueousmedium is 5-9.
 10. A kit comprising a means for storing a composition asclaimed in claim 1, and such a composition contained in said storagemeans.
 11. A method of making a storage stable composition comprising amatrix carrying reactive electrophilic squaryl groups, said methodcomprising i) reacting a matrix comprising primary and/or secondaryamine groups with a bifunctional nucleophilic squaric acid derivativethereby introducing squaryl groups on the matrix to give an activatedmatrix ii) washing the activated matrix with an aqueous medium; and iii)storing said activated matrix in said aqueous washing medium for atleast a week.